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. 2016 Dec 22;7:13853. doi: 10.1038/ncomms13853

Figure 4. PKCɛ and Aurora B influence CHMP4C localization during cytokinesis.

Figure 4

(a) DLD1 GFP-Aurora B cell lines were transiently transfected with three separate CHMP4C siRNA, imaged using live-cell time-lapse microscopy and scored for outcome of cytokinesis; successful cytokinesis resulting in two daughter cells or failed cytokinesis resulting in a binucleate cell. Graph represents the mean (±s.e.m.) of three independent experiments; a minimum of 50 cells were counted per experiment. Two-way analysis of variance (ANOVA), **=P≤0.01, ***=P≤0.001. (b) HeLa cells were transfected with PKCɛ siRNA, a pool of three CHMP4C siRNA or PKCɛ siRNA and the pool of CHMP4C siRNA, imaged using live-cell time-lapse micrsocopy and scored for outcome of cytokinesis as per the criteria above. Graph represents the mean (±s.e.m.) of three independent experiments; a minimum of 50 cells were counted per experiment. Two way ANOVA, not significant (NS)=P>0.05, **=P≤0.01. (c) DLD1 GFP-Aurora B WT and S227A cell lines (green) were transiently transfected with HA-CHMP4C (red) to look for midbody localization during cytokinesis. Cells were scored for the presence or absence of HA-CHMP4C at the midbody. A minimum of 12 high-resolution, single-cell images per condition from four experiments were acquired; a representative image is shown here. Scale bar, 10 μm. Student's t-test, *=P≤0.05. (d) DLD1 GFP-Borealin WT and S165A cell lines (green) were transiently transfected with HA-CHMP4C (red) to look for midbody localization during cytokinesis. Cells were scored for the presence or absence of HA-CHMP4C at the midbody. A minimum of five high-resolution, single-cell images per condition from two experiments were acquired; a representative image is shown here. Scale bar, 10 μm. (e) Working model: chromatin trapped in the cytokinesis furrow engages the Aurora B-dependent abscission checkpoint. We propose that the association through T232 only phosphorylated Aurora B and Borealin, CHMP4C is maintained in a S210 phosphorylated, closed, inactive conformer distal to the midbody. On bridge resolution, PKCɛ phosphorylates Aurora B on S227, which in turn phosphorylates Borealin S165, allowing for CHMP4C to assume an open, active conformation to polymerize with other ESCRT-III components and facilitate successful abscission.